1. Field of the Invention
The present invention relates to a method of detecting bacterial contamination in platelets or red blood cells using automated epifluorescence microscopy.
2. Description of the Prior Art
Bacterial contamination in blood platelets occurs in about 1 in every 1000-2000 units. Nationwide, this produces at least 150 cases per year of severe illness and occasional deaths. Because of this, there is a need for a rapid, inexpensive method which can detect and count bacteria in platelets at concentrations below 105 colony forming units (CFU)/ml. To this date, only culturing, polymerase chain reaction (PCR), and fluorescence conjugated antibiotics are consistently able to detect bacteria at concentrations below 105 colony forming units (CFU)/ml. The automated culture method, which can detect a single organism, requires at least 24 hrs to make a determination. PCR works well down to 104 CFU/ml but is labor intensive and can take several hours. The use of fluorescent antibiotics has only been tested on one bacterial strain.
Fluorescent dyes are routinely used in microbiology to detect proteins, amino acids, nucleic acids, and whole cells, as well as biological activity in a wide variety of systems. U.S. Pat. No. 4,693,972 to Mansour et al. discloses a method for detecting microorganisms in blood based on lysis of the blood components and staining the microorganisms in the blood with the fluorescent dye ethidium bromide. However, because ethidium bromide is not membrane-permeable, it does not stain live bacteria. Thus, the sample preparation requires an extra step of permeablizing the bacterial cell membrane without disrupting the cell if detection of bacterial contamination is desired. Mansour et al. use a cytometer to count the microorganism cells and a centrifuge to concentrate microorganisms in the sample before it is analyzed. Cytometers are expensive and their operation is labor intensive.
U.S. Pat. No. 5,798,221 to AEgidius uses ethidium bromide to stain bacteria in a milk sample followed by counting the bacterial cells by passing the sample through a cytometer. Due to the poor permeability of ethidium bromide into bacterial cells, a combination of a chelating agent and a detergent is required in order to digest protein particles and enhance the staining of bacterial cells with ethidium bromide.
U.S. Pat. No. 4,717,660 to Schulte discloses a method for detecting microorganisms in a blood sample involving: a) selectively staining the microorganisms in the blood sample using a fluorochrome dye such as ethidium bromide or acridine orange and a staining buffer, b) centrifuging the sample with a centrifuge tube provided with a float, and c) detecting the fluorescence using flow cytometry. Again, using flow cytometry is expensive and labor-intensive. In addition, a staining buffer is required to enable the staining of microorganisms using ethidium bromide. Acridine Orange also readily stains other particles including platelets. The method of Schulte has difficulty in detecting bacterial contamination in platelets at concentrations below 105 CFU/ml.
U.S. Pat. No. 5,828,716 to Bisconte de Saint Julien discloses an automated method for analyzing particles at magnifications below 100xc3x97 magnification. At such a low magnification, the size and shape information critical to distinguishing bacteria from other fluorescent particles is lost.
U.S. Pat. No. 5,545,535 to Roth et al. discloses a method of analyzing a sample thought to contain bacteria using an aqueous solution comprising one or more fluorescent dyes. Roth et al. further discloses several generic methods for detecting the stained bacteria including the use of epifluorescence microscopy coupled with digital image acquisition. Roth et al. also exemplifies the use of filtration to concentrate the bacteria-containing samples. However, the lowest bacterial concentration detected in Roth et al. is 5xc3x97105 CFU/ml due to the limitations of its methods.
Therefore, it is an objective of certain embodiments of the present invention to provide a method for detecting bacteria which can detect bacteria at concentrations as low as 3.0xc3x97103 CFU/ml, which is below the clinically significant level.
It is another objective of certain embodiments of the present invention to provide a method for detecting bacteria which does not require a culturing step so as to provide rapid detection.
It is a further objective of certain embodiments of the present invention to provide a method for detecting bacteria in platelet-containing sample which is not labor intensive and has the capability of distinguishing a variety of sources of fluorescence from that of bacteria by taking advantage of digital image acquisition technology.
In a first aspect, the present invention relates to a method for ascertaining the presence of bacteria in a sample containing platelets or red blood cells comprising the steps of: lysing a substantial portion of the platelets or red blood cells in the sample; staining at least a substantial portion of the bacteria using a membrane-permeable nucleic acid stain; filtering the sample using a membrane filter to obtain a material containing substantially all of the stained bacteria; and analyzing the filtered material using epifluorescence microscopy to ascertain the presence of the bacteria in the sample.
In a second aspect, the present invention relates to a method for determining the concentration of bacteria in a sample containing platelets or red blood cells comprising the steps of: lysing a substantial portion of the platelets or red blood cells without destroying a substantial amount of the bacteria in the sample; staining the bacteria using a membrane-permeable nucleic acid stain; filtering the sample using a membrane filter to obtain a material containing substantially all of the stained bacteria; and analyzing the material using epifluorescence microscopy and digital image acquisition and analysis to determine the concentration of the bacteria in the sample.